ABSTRACT Abnormal separation of the respiratory system from the foregut leads to the common birth defect esophageal atresia/tracheoesophageal fistula (EA/TEF) which affects 1/2,500-3000 newborns. Although the anomalycanbecorrectedwithsurgicalintervention,upto72%ofsurvivingadolescentsandadultscontinueto suffer from respiratory problems throughout their lifetime, suggesting a connection between EA/TEF and lung abnormalities. Consistently, EA/TEF is always accompanied by abnormal lungs (e.g. lobe fusion) in animal models,althoughtheunderlyingmechanismisunknown.Werecentlyshowedthatanepithelialsaddleformed at the lung-esophageal boundary moves upward to split the lung and trachea from the esophagus. However, severalimportantquestionsremaintobeanswered.Howisthelunginvolvedinsaddleformationandmovement? Whatistheunderlyingcellularandmolecularmechanism?Weaimtouseacombinationoforganculture,frog, and mouse models to address these issues. Our lineage tracing data show that derivatives of respiratory progenitorcells(Nkx2.1positive)integrateintotheesophagusduringseparation.Moreover,ourpreliminarydata suggest that a unique lung epithelial progenitor subpopulation (Sox2;?Sox9;?Isl1 positive) located at the lung- esophageal boundary plays critical roles in the formation of the saddle. We further found that the loss of the transcription factor Sox2 or Isl1 in the lung progenitors, including the subpopulation, leads to EA/TEF and abnormal lungs in both frogs and mice. Interestingly, these abnormalities are accompanied by a reduction of extracellularmatrix(ECM)proteinsincludingFrasfamilymembersFras1andFrem2whichareknowntoregulate lung development. We therefore hypothesize that the Sox2/Isl1 axis regulates ECM proteins in a lung epithelialprogenitorsubpopulation(Sox2;?Sox9;?Isl1positive)thatisrequiredforrespiratory-esophageal separationandlungdevelopment.Wewilltestthehypothesiswiththreespecificaims:Aim1todeterminethe contributionofthelungepithelialprogenitorsubpopulationtothesaddleformationandrespiratory-esophageal separation;?Aim2totestthehypothesisthatSox2regulatesIsl1inthelungepithelialprogenitorsubpopulation to control respiratory-esophageal separation;? Aim3 to test the hypothesis that Isl1 regulates the separation process and lung development through ECM proteins. Notably, chromosomal deletion of the region covering ISL1 (and other genes) has been found in patients with EA/TEF. Our findings therefore will provide direct evidence and mechanistic insight into the role of Sox2/Isl1/ECM axis in the pathogenesis of this defect and associatedlungabnormalities.